The commercial scale purification of various therapeutic biomolecules, such as monoclonal antibodies, is currently accomplished using bead-based chromatography resins. Monoclonal antibodies continue to gain importance as therapeutic and diagnostic agents. The process of screening hybridoma libraries for candidate mAbs is both time consuming and labor intensive. Once a hybridoma cell line expressing a suitable mAb is established, a purification methodology must be developed to produce sufficient mAb for further characterization. A traditional method for purifying involves using Protein A or Protein G affinity chromatography, as well as ion exchange chromatography. The purified antibody is desalted and exchanged into a biological buffer using dialysis. The entire process typically requires several days to complete and can be particularly onerous if multiple mAbs are to be evaluated in parallel.
Chromatography resins are currently prepared with various ligand structures that enable the beads to function in affinity, cation-exchange, or anion-exchange modes. These resins demonstrate a high porosity and large surface areas that provide materials with sufficient adsorptive capacities for the batch processing of biomolecules at production scales (e.g., 10,000 liters). Chromatography resins typically present a spherical structure that enables an efficient column packing with minimal flow non-uniformities. The interstitial spaces between the beads provide flow channels for convective transport through the chromatography column. This enables chromatography columns to be run with large bed depths at a high linear velocity with a minimal pressure drop. The combination of these factors enables chromatography resins to present the required efficiency, high permeability, and sufficient binding capacity that are required for the large-scale purification of biomolecules.
In bead-based chromatography, most of the available surface area for adsorption is internal to the bead. Consequently, the separation process is inherently slow since the rate of mass transport is typically controlled by pore diffusion. To minimize this diffusional resistance and concomitantly maximize dynamic binding capacity, small diameter beads can be employed. However, the use of small diameter beads comes at the price of increased column pressure drop. Consequently, the optimization of preparative chromatographic separations often involves a compromise between efficiency/dynamic capacity (small beads favored) and column pressure drop (large beads favored).
Chromatography media typically has a very high cost (>$1000/L) and significant quantities are required for large scale production columns. As a result, biopharmaceutical manufacturers recycle chromatography resins hundreds of times. Each of these regeneration cycles consumes substantial quantities of buffer media, and each step incurs additional costs associated with the validation of each cleaning, sterilization, and column packing operation.
Several technologies are described in the patent literature and marketed commercially for biopharmaceutical separations based on functionalized fibrous media and/or composites. Most rely on incorporating a porous gel into the fiber matrix, the gel providing the needed surface area to gain reasonable binding capacities. However, in such constructions, poor uniformity in gel location and mass generally leads to poor efficiencies (shallow breakthrough and elution fronts). In addition, resistance to flow can be high even for short bed depths, a problem often aggravated by gel compression under modest pressure loads. Another approach taken has been the incorporation of particulates within the fiber matrix, the particulates often porous and possessing a native adsorptive functionality, examples being activated carbon and silica gel.
Recently, EMD Millipore has developed a fiber based chromatography media for biomolecule purification applications that utilizes a surface-functionalized winged fiber as the adsorptive media. The winged projections on the fiber surface afford a much higher surface area than ordinary round fibers of similar dimensions. The resulting surface functionalized fiber media also has a much higher protein binding capacity than similarly functionalized fibers which lack such winged projections.
Other emerging technologies are currently in development for protein purification applications and these include membrane adsorbers, monoliths, and flow-through adsorber purification methods using commercial resin systems. While membrane adsorbers and monoliths may provide acceptable binding capacities for these applications, these technologies typically have their own scale limitations and the extremely high cost of such purification media may further limit their adoption into a price sensitive industry with an existing purification process template.